Multimodal Mass Spectrometry Imaging of N-Glycans and Proteins from the Same Tissue Section

Abstract

On-tissue digestion matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) can be used to record spatially correlated molecular information from formalin-fixed, paraffin-embedded (FFPE) tissue sections. In this work, we present the in situ multimodal analysis of N-linked glycans and proteins from the same FFPE tissue section. The robustness and applicability of the method are demonstrated for several tumors, including epithelial and mesenchymal tumor types. Major analytical aspects, such as lateral diffusion of the analyte molecules and differences in measurement sensitivity due to the additional sample preparation methods, have been investigated for both N-glycans and proteolytic peptides. By combining the MSI approach with extract analysis, we were also able to assess which mass spectral peaks generated by MALDI-MSI could be assigned to unique N-glycan and peptide identities.

MALDI Mass Spectrometry Imaging of N-Linked Glycans in Cancer Tissues

“…The basic approach is to spray a molecular coating of the enzyme that releases N-linked carbohydrates from carrier proteins, peptide N-glycanase (PNGaseF), on-tissues, followed by matrix application and MALDI-IMS. Beyond the initial publications, eight other reports from multiple laboratories have been published for FFPE cancer tissues [Powers et al, 2015; Drake et al, 2015; Holst et al, 2016; Heijs et al, 2016; Everest-Dass et al, 2016] and other non-cancer FFPE tissue types [Briggs et al, 2016; Toghi Eshghi et al, 2014; Gustaffson et al, 2015]. Compared to other biomolecules targeted by MALDI-IMS like lipids, metabolites and proteins/peptides, IMS of N-glycans has some unique analytical aspects.…”

Recent Advances in the Mass Spectrometry Methods for Glycomics and Cancer

“…Human formalin-fixed paraffin-embedded (FFPE) tissues have been used to characterize the spatial profile and localization of released N-glycans using MALDI-Imaging Mass Spectrometry (MALDI-IMS). 45–46 FFPE tissues collected from human hepatocellular carcinomas, prostate, and pancreas have been found to have distinct glycan profiles when compared to adjacent non-tumor tissue slices. 45, 47 For validation of alterations in glycan expression, tissue microarrays incorporating multiple FFPE tumor and normal tissue cores have been used in combination with MALDI-IMS.…”

Mass Spectrometry Imaging: A Review of Emerging Advancements and Future Insights

“…46,50 More recently, researchers have been able to extract metabolites and glycans. 47,51 With more standardized protocols, the extensive FFPE samples available will be utilized more readily for MSI workflow, allowing for exciting possibilities to examine many clinical specimens and a flood of new information to help guide researchers in future endeavors.…”

“…3 In particular, the development of on-tissue digestion with trypsin has increased protein coverage by allowing not only higher molecular weight species to be analyzed but also by letting other more sensitive, higher resolution instrumental platforms become available for protein analysis. 9,47 Animal tissues are not the only sample types to have benefitted from this technique, as plant peptides have been imaged to compare those found in seedling and mature Medicago truncatula . 209 Alternative ionization techniques beyond MALDI have been applied to better image proteins and peptides.…”

“…As discussed previously, on-tissue digestion sequentially with PNGaseF and trypsin enabled MSI analyses of both released N-glycans and tryptic protein fragments on a single tissue. 47 It should be noted though that native glycan imaging has been performed. 211 N-glycans have been analyzed in variety of samples, including kidney, bone, and cancerous tissues.…”